Bridging the gap: bispecific proteins for targeted radiotherapy

From the Orozco Laboratory, Clinical Research Division

Radiation is a common and effective therapy modality for acute myeloid leukemia (AML). However, intensive therapies such as total body irradiation can be too harsh to be tolerated in many cases, including in elderly populations, who make up the majority of AML patients. One strategy to reduce toxicities associated with radiation therapy, called radioimmunotherapy (RIT), involves the use of antibodies to deliver radioactive isotopes specifically to tumor cells, thereby sparing healthy tissues. A major drawback of directly conjugating long-lived antibody molecules to radioisotopes is the exposure of non-targeted tissues to radiation while the drug conjugate circulates through the body in search of its target. Thus, two-step pre-targeted RIT (PRIT) solutions have been developed, in which the cancer-targeted antibody is delivered prior to the radioisotope. Dr. Johnnie Orozco, from the Fred Hutch Clinical Research Division, and colleagues investigated the use of bispecific fusion proteins to bridge the gap between the leukemia and the radioisotope as a novel PRIT modality against AML. Their findings were recently published in Molecular Cancer Therapeutics.

An established PRIT method involves the use of the extraordinary binding affinity between streptavidin (SA) and biotin molecules, in which SA-conjugated antibodies (Ab-SA) are first administered to patients, followed later by a biotinylated radioisotope chelate (90Y-DOTA-biotin). DOTA is an organic compound that encapsulates, or chelates, the Yttrium atom to make it more soluble.  In this setting, the larger and more stable Ab-SA accumulates on the target cells before administration of 90Y-DOTA-biotin. The smaller 90Y-DOTA-biotin is then administered and quickly circulates throughout the body, binding to Ab-SA at the target site. Any unbound 90Y-DOTA-biotin is rapidly excreted from the body, minimizing radiation exposure to healthy tissues. However, several problems with this strategy, including poor penetration of tumors by the large antibody conjugates, interference by naturally occurring biotin molecules in the body, and immunogenicity of SA-conjugates, could limit its effectiveness. Thus, Orozco and colleagues developed novel bispecific constructs that bind to both the CD45 leukemia antigen and the radiolabeled ion chelate 90Y-DOTA and tested their efficacies against animal models of leukemia.

Bispecific fusion proteins for targeted radiotherapy.
Model of anti-leukemia bispecific antibodies. Figure provided by Dr. Orozco.

The authors produced both mouse- and human-specific CD45 versions of their fusion molecules. The first iteration of their molecule involved the fusion of a full-length mouse CD45-specific antibody (or non-targeting control) with a smaller synthetic antibody fragment, called a single-chain variable fragment (scFv), that binds to 90Y-DOTA. After confirming that their antibody fusion molecule could successfully target 90Y-DOTA to mouse leukemia targets in vitro, they moved into a mouse model of AML to confirm proper targeting. Encouragingly, they observed preferential accumulation of the radioisotope in the spleen and bone marrow leukemia target tissues, with lower uptake in non-target organs. In a therapeutic setting, they observed a significant extension of survival in mice receiving the CD45-targeted bispecific antibody compared to the non-targeting control bispecific antibody, establishing therapeutic benefit for this treatment modality.

Orozco et al. next produced a more compact version of their bispecific molecule by fusing both the 90Y-DOTA-specific scFv and a human CD45-specific scFv (or non-targeting control) to an Fc fragment hinge. Using this molecule, they established faster clearance from the circulation of healthy mice of the bispecific molecule than a SA-conjugated anti-CD45 scFv, indicating a potential benefit of the bispecific molecule of avoiding non-specific binding associated with SA-conjugates. Using a xenograft model of human AML, these bispecific molecules exhibited preferential accumulation in leukemia target tissues, and a survival benefit over the non-targeting control treatment. Importantly, while these studies were not powered to discriminate between the benefits of SA-conjugated PRIT and bispecific molecule-based PRIT therapies, animals treated with either SA-conjugated PRIT or bispecific molecule-based PRIT exhibited comparable survival extension, though bispecific molecule-based PRIT appeared to confer better tumor size control in this solid tumor setting.

“Our studies with preclinical leukemia models suggest that bispecific antibody constructs targeting CD45 and Y-DOTA are effective therapeutic agents for myeloid leukemia,” explain the authors. This work establishes bispecific molecules as feasible alternatives to SA-conjugated PRIT. Further studies will be required to establish whether bispecific molecule-based PRIT can confer superior therapeutic benefit in certain cancer settings compared to SA-conjugated PRIT. “One option to improve leukemia outcomes might be to strategically employ PRIT approaches in the appropriate disease setting,” note the authors. This summer Dr. Orozco and the RIT team were awarded an NCI R37 grant to evaluate possible synergy of these bispecific antibodies in combination with novel AML targeted agents, which should build the foundation for future clinical trials. Dr. Orozco noted, “How to best deploy these therapies will require the epitome of ‘team science’, and we’re fortunate that The Hutch naturally brings together the radioimmunotherapy investigators from the Hutch, the radiochemists from the UW, and leukemia investigators from the SCCA.”

This work was supported by the National Cancer Institute, the National Institutes of Health, the American Society for Blood and Marrow Transplantation, the Robert Wood Johnson Foundation / ASH Amos Medical Faculty Development Program, the David and Patricia Giuliani Foundation, and the Frederick Kullman Foundation.

UW/Fred Hutch Cancer Consortium members Johnnie Orozco, D. Scott Wilbur, Damian Green, Ajay Gopal, Brian Till, and Brenda Sandmaier contributed to this work.

Orozco JJ, Kenoyer AL, Lin Y, O'Steen S, Guel R, Nartea ME, Hernandez AH, Hylarides MD, Fisher DR, Balkin ER, Hamlin DK, Wilbur DS, Orcutt KD, Wittrup KD, Green DJ, Gopal AK, Till BG, Sandmaier B, Press OW, Pagel JM. Therapy of Myeloid Leukemia using Novel Bispecific Fusion Proteins Targeting CD45 and 90Y-DOTA. Mol Cancer Ther. 2020 Dec;19(12):2575-2584. doi: 10.1158/1535-7163.MCT-20-0306.